With the advent of recombinant DNA methodology and especially in view of its evident commercial applicability, the ability to synthesize oligodeoxyribonucleotides of defined sequences has become increasingly important.
As now is very well recognized, RNA and DNA are polynucleotides referred to as nucleic acids. The polynucleotides, in turn, are composed of monomers (nucleotides). A nucleotide is a phosphate ester of the N-glycoside of a nitrogenous base and consists of a purine or a pyrimidine base, a pentose (D-ribose in RNA or 2'-deoxy-D-ribose in DNA), and a phosphate group.
Four nitrogenous bases are present in both DNA and RNA. The four present in DNA are: ##STR1## The nitrogenous bases in RNA differ from those in DNA only in that uracil (U) replaces thymine. ##STR2##
The combination of a nitrogenous base at the point of the asterisk (*) in the foregoing formulas with a ribose at its 1'-position is called a ribonucleoside (D-ribose) or a deoxyribonucleoside (2'-deoxy-D-ribose). The corresponding ribonucleotide or deoxyribonucleotide is produced by addition of a phosphate group at the 3'-position of the ribose.
The thus-defined suitably-blocked ribonucleotide or deoxyribonucleotide represents the basic building block in the synthesis of RNA or DNA, respectively. A standard and highly attractive method for synthesizing RNA or DNA is known in the literature as the "triester method". Using the synthesis of a polydeoxyribonucleotide as an example, the procedure involves coupling a mononucleotide or oligonucleotide having a 3'-phosphate diester with a mononucleoside, a blocked 3'-hydroxy oligonucleotide, a mononucleotide, or an oligonucleotide having an available 5'-hydroxyl group. This method can be represented schematically as follows: ##STR3## In the foregoing, B is a nitrogenous base selected from adenine, cytosine, guanine, and thymine, each of the first three having their reactive moieties blocked by suitable protecting groups; R is a blocking group for the 5'-hydroxyl; R.sup.3 is a blocking group for the 3'-hydroxyl cleavable under alkaline conditions or a group of the formula ##STR4## and R.sup.1 and R.sup.2 are selectively removable groups which block the reactive phosphate moiety.
Additional discussion of the triester method can be found in various publications including, for example, Narang, S. A., Hsiung, H. M., and Brousseau, R., "Improved Phosphotriester Method for the Synthesis of Gene Fragments", Methods in Enzymology, Vol. 68, Academic Press, New York, N.Y., (1979), pp. 90-98; and Narang, S. A., Brousseau, R., Hsiung, H. M., and Michniewicz, J. J., "Chemical Synthesis of Deoxyoligonucleotides by the Modified Triester Method", Methods in Enzymology, Vol. 65, Academic Press, New York, N.Y., (1980), pp. 610-620.
The triester method described above, of course, has been applied in coupling two oligonucleotides, an oligonucleotide and a mononucleotide, or, as specifically illustrated above, two mononucleotides. Whatever the entities, the reaction involves the coupling of an available 5'-hydroxyl with a 3'-phosphate diester group. Moreover, the reactant containing the available 5'-hydroxyl can have a blocked terminal phosphate or such can be lacking (i.e., R.sup.3 is a 3'-hydroxyl blocking group). The two reactants are coupled in the presence of a solvent, typically pyridine, and in the presence of a coupling agent, for example, 2,4,6-trimethylbenzenesulfonyl tetrazolide.
Whatever the structure of the particular nucleotide reactants, it is essential that the reactant having an available 5'-hydroxyl group be fully blocked at the terminal 3'-phosphate group if such is present. The group R.sup.2 appearing in the foregoing formulas represents a blocking group that completes protection of the terminal 3'-phosphate group. If further coupling of the product polynucleotide at its 3'-phosphate site is intended, it is important that the R.sup.2 group be one that is removable without disruption of other blocking groups on the molecule.
Customarily, the R.sup.2 group of choice is .beta.-cyanoethyl, a group which is selectively removable under mild alkaline conditions. A highly preferred method for selectively removing a .beta.-cyanoethyl group from a nucleotide or oligonucleotide uses anhydrous triethylamine-pyridine. This method is reported in Adamiak, R. W., Barciszewska, M. Z., Biala, E., Grzeskowiak, K., Kierzek, R., Kraszewski, A., Markiewicz, W. T., and Wiewiorowski, M., Nucleic Acids Research 3, 3397-3408 (1976), and Sood, A. K., and Narang, S. A., Nucleic Acids Research 4, 2757-2765 (1977). Although this method has been highly successful in achieving selective elimination of the .beta.-cyanoethyl blocking group, it suffers from the fact that it is time-consuming, taking 4-6 hours to complete (see Sood et al., supra, p. 2758).
It now has been discovered that the selective removal of a .beta.-cyanoethyl blocking group can be achieved much more rapidly, i.e., in a matter of a few minutes, using diethyl amine. It is to such a process that this invention is directed.